PB1753: EVALUATION OF IMMUNOPHENOTYPIC CHARACTERISTICS OF RESIDUAL CELLS IN COMPARISON WITH NORMAL HEMATOGONES AND MATURE CELLS IN CHILDREN WITH B-CELL LYMPHOBLASTIC LEUKEMIA
Authors: Oksana Karnabeda, Kateryna Vilchevska, Iryna Bakhchivandzhi
Journal: HemaSphere (2023)

Topic: 2. Acute lymphoblastic leukemia - Clinical Background: Despite some experience gained so far in the use of flow cytometry for monitoring MRD (Minimal Residual Disease), the development of new markers and improvement of the methodology for MRD detection seems to be promising. Aims: Evaluation of changes in the level of markers expression during protocol therapy in children with B-cell lymphoblastic leukemia when determining minimal residual disease (MRD). Methods: The study included children (n=26: 14 boys and 12 girls aged from 1 month to 16 years) with the B-cell variant of acute lymphoblastic leukemia. The diagnosis of pro-B-CLL, common-B-CLL and pre-B-CLL was established according to the WHO classification (EGIL-95) and based on the assessment of the immunophenotype of tumor cells by flow cytometry. Stratification of patients into risk groups and antitumor therapy was performed according to the ALLIC-BFM 2002 protocol. The material for analysis was bone marrow. The method of 3- and 2-color staining was used for all samples. Dye-conjugated antibodies were used: fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP). Antibody-producing clones were carefully selected for their reactivity and absence of background (nonspecific) staining. All antibodies were obtained from BD Biosciences: CD45, CD14, CD5, CD10, CD19, CD34, HLA-DR, CD3, CD7, CD22, CD11C, CD15, CD117, CD38, CD20, CD13, CD4, CD8, CD58, CD3, cytIgM, sIgM, cytCD22, TdT, cytCD79a MPO. The stained samples were analyzed on a FACScalibur flow cytometer (BD Biosciences) using Cell Quest software (BD Biosciences). Mean Fluorescence Intensity (MFI) values were used to compare expression levels. The Statistica 6.0 program was used for statistical processing of the results. Results: Immature cells (blasts and hematogones) differ from mature lymphocytes by lower CD45/SSC parameters. These parameters change depending on the degree of maturation. Heterogeneity of antigen expression on blast cells: • 10.8% of cases - heterogeneous expression of CD10; • 30.6% of cases - heterogeneous expression of CD34; • 10.6% of patients - blasts turned out to be CD34-negative; • 44% - all blasts co-expressed CD 10 and CD34; • in 4% of patients, tumor cells did not express either CD10 or CD34. Despite clear differences between tumor and normal cells in the expression of various antigens, no single universal marker has been identified that would be acceptable for monitoring MRD in all cases of B-CLL. Summary/Conclusion: • Monoclonal antibodies CD10, CD19, but not CD34 can be used to distinguish residual tumor cells from normal progenitors during MRD monitoring on the 15th day of therapy. • Most blast cells in B-CLL express CD34 antigen on their surface. For CD34-positive B-CLL, when isolating the B-lymphoblastic population among the elements of hematopoiesis (during regeneration), it is possible to use indicators of the fluorescence intensity of the CD34 marker in combination with CD19/SSC, which will allow in this subgroup of B-CLL to clearly identify residual B-lymphoblasts and thus, to avoid false conclusions when assessing remission. Keywords: Minimal residual disease (MRD), Immunophenotype, B cell acute lymphoblastic leukemia